PROJECT SUMMARY The goal of this research proposal is to dissect the novel regulatory circuits and genes underlying novel traits, to get a better understanding of the genetic basis of morphological and cellular innovation. Every morphological structure or trait originated at some time point in the past and evolved under various evolutionary paths. However, it is unknown how a novel trait originates and how gene and regulatory networks spatially orchestrate the development of the novel cell types, tissues, and organs. Identifying the processes driving and governing morphological and functional diversity and complexity is a major step towards understanding the evolution of complex life. However, our understanding of this process is still limited. The long-term goal of this research program is to functionally characterize the molecular genetic basis of novel cell clusters and novel morphological phenotypes. The central hypothesis is that evolutionary innovations emerging from novel regulatory networks depend on changes in transcription factors and enhancers. Guided by preliminary data including single cell RNA sequencing and well-established theories, the proposed research will test the central hypothesis using an integrative approach. We will determine: 1) regulatory network innovation in novel cell clusters in Drosophila, 2) enhancers responsible for transcription factor expression changes and downstream expression network modification, and 3) the genetic regulatory basis of a novel trait. We performed single-cell sequencing and RNA fluorescent in situ hybridization (FISH) on testis and found a novel cell cluster differentiated between Drosophila species. Combined with ATAC-sequencing data, we will use modeling and functional studies to study the genetic basis of the novel cell cluster. Following this hypothesis that novel enhancers of transcription factors (TFs) are essential for novel traits, we will study the cause of a recurrent novel trait and test the hypothesis that a novel regulatory circuit is essential for a novel trait. We hypothesize that novel enhancers or cis-regulatory motifs of TFs are essential for whole-genome level changes in chromatin accessibility. To test it, we will identify enhancer and motif changes between closely related species to provide insights into enhancer and TF binding affinity co- evolution. This study will provide important insights into the evolution of transcription regulatory networks and their contributions to novel morphological and cellular traits. Altogether, our integrative approach will help to elucidate the origination and evolution of novel regulatory circuits and their contributions to phenotypic innovation.